A. Field of the Invention
The present invention relates generally to the fields of biochemistry and molecular biology. More particularly, it relates to lipid metabolism and the regulation of lipid metabolism. In a specific embodiment, it relates to inhibition of intestinal absorption of cholesterol by ligands that bind to the retinoid X nuclear hormone receptor (RXR).
B. Description of Related Art
Sterols are structural lipids present in the membranes of most eukaryotic cells. These lipids are rigid and characterized by a four ring hydrocarbon steroid nucleus. Sterols are required not only to impart membrane fluidity, but also serve as the precursors for a variety of products with specific biological activities. For example, cholesterol, an amphipathic sterol with a polar hydroxyl head group and nonpolar hydrocarbon body (the steroid nucleus), is the major sterol found in animal tissues. Cholesterol is an essential molecule, playing a critical role in the structural integrity of cell membranes, a precursor for steroid hormones and serves as a precursor for bile acids. Cholesterol is synthesized in the liver from isoprene precursors and further obtained via dietary intake.
Although cholesterol is a requisite molecule, high levels of blood cholesterol or hypercholesterolemia has been implicated in atherosclerosis, heart attack, and stroke (Schultheis, 1990; Mitchell, 1990). Hypercholesterolemia, if not controlled, is one of several conditions that can lead to coronary artery disease. Coronary artery disease is the leading cause of death in the United States, accounting for approximately 600,000 deaths per year. Thus, the need exists for methods of treatment that can reduce cholesterol levels and methods to screen patients at risk for high cholesterol.
Possible targets for treatment are transcription factors involved in cholesterol metabolism. One such set of factors, nuclear receptors, are ligand-activated transcription factors that govern aspects of every major developmental and metabolic pathway (reviewed in Kastner et al., 1995; Mangelsdorf et al., 1995). For example, the LXRs were first identified as xe2x80x9corphanxe2x80x9d members of the nuclear receptor superfamily whose ligands and functions are unknown (Willy and Mangelsdorf, 1998). The LXRs have recently been shown to be activated by a specific class of naturally occurring, oxidized derivatives of cholesterol, including 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, and 24,25(S)-epoxycholesterol (Janowski et al., 1996; Lehmann et al., 1997). Oxysterols are concentrated in tissues where cholesterol metabolism and LXR expression are high, such as liver, brain, and placenta (Lavy et al., 1977; Spencer et al., 1985; Lxc3xctjohann et al., 1996).
LXRs function as heterodimers with the retinoid X receptors (RXRs), and thus, the RXR/LXR complex can be activated by both RXR ligands (i.e., rexinoids) and oxysterols (Teboul et al., 1995; Willy et al., 1995; Janowski et al., 1996). Two LXR proteins (xcex1 and xcex2) are known to exist in mammals. The expression of LXRxcex1 is restricted, with highest levels in the liver (hence, the name liver X receptor) and lower but significant levels in kidney, intestine, spleen, and adrenals (Apfel et al., 1994; Willy et al., 1995). LXRxcex2 expression is more widespread and has been found in nearly every tissue examined (Shinar et al., 1994; Song et al., 1994).
The pattern of expression of LXRs and their oxysterol ligands first suggested that these receptors may have a role in cholesterol metabolism. Cholesterol has two essential metabolic fates in mammals: conversion into steroid hormones or bile acids. Since steroid hormone synthesis is known to be governed by the orphan nuclear receptor steroidogenic factor-1 (SF-1) (Parker and Schimmer, 1997), it is possible that LXRs are involved in bile acid synthesis (Janowski et al., 1996). A likely target for any bile acid inducer is cholesterol 7xcex1-hydroxylase (Cyp7a), the rate-limiting enzyme in the classical bile acid synthesis pathway (Janowski et al., 1996; Lehmann et at., 1997). Experiments by the inventors and others have shown that the Cyp7a promoter contains a functional LXR response element that can be activated by RXR/LXR heterodimers in an oxysterol- and retinoid-dependent manner (Lehmann et (al., 1997). The formation of bile acids is one of two major pathways for the catabolism and excretion of cholesterol in mammals (Russell and Setchell, 1992). Perturbations in this pathway may lead to a variety of disorders, including cholesterol gallstones, atherosclerosis, and some lipid storage diseases (Akiyoshi et al., 1986; Turley and Dietschy, 1988; Carey and Duane, 1994). Together, these observations have raised an interesting possibility that LXRs may function as transcriptional control points in bile acid metabolism.
The RXR protein of RXR homo- and heterodimers has been observed to be regulated by 9-cis retinoic acid, which binds to the carboxy-teminus of RXR (Mangelsdorf and Evans, 1995). RXR can form heterodimers with numerous other proteins in the nuclear receptor superfamily, including LXR. Depending on the receptor protein that dimerizes with RXR, and the ligands present, the resulting effects of the heterodimer on transcription can vary. Synthetic retinoids have been found to selectively bind and activate RXRs (U.S. Pat. No. 5,780,676 and U.S. Pat. No. 5,455,265).
The potential to modulate lipid concentrations in vivo, by targeting proteins of the nuclear hormone receptor superfamily with specific ligands would be particularly useful in the treatment of various diseases related to lipid metabolism. For example, high blood cholesterol levels are associated with coronary disease. Lowering dietary cholesterol intake can significantly reduce cholesterol levels in most people. However, lowering dietary intake of cholesterol often is not enough, as certain individuals sustain high cholesterol blood levels due to inefficient endogenous cholesterol homeostasis. Thus, the ability to reduce blood cholesterol levels would prove to be extremely beneficial to these individuals. Currently, there are various drugs that are administered to treat hypercholesterolemia and other abnormal blood lipid levels. For example, cholestyramine and colestipol are resins that bind bile acids in the intestinal tract, causing the liver to increase its production of bile acids and thus lower the cholesterol levels, by converting cholesterol into bile acid. However, the efficacy of these drugs is low, they are unpleasant to take, and result in constipation and bloating. Nicotinic acid, gemfibrizol, probucol, and lovastatin also are drugs used to lower blood lipid levels, but each has undesirable side effects associated with it. For example, probucol lowers total cholesterol levels, but also results in the undesirable lowering of HDL cholesterol.
Given the high incidence of coronary artery disease in the United States and its association with high cholesterol, there is a high demand for a treatment that can lower cholesterol levels without adverse side effects. Furthermore, a treatment that can lower LDL cholesterol levels, without affecting total lipid levels is highly desirable.
It is, therefore, a goal of the present invention, to provide both compositions and methods that are related to cholesterol metabolism and abnormalities therein. More particularly, the invention is intended to provide tool for the identification of compositions for use in treating cholesterol-related pathologies, as well as therapeutic methods of use.
Thus, in a first embodiment, the present invention provides a non-human transgenic mammal, the cells of which comprise at least one non-functional endogenous LXRxcex1 allele. The transgenic mammal may further comprise two non-functional endogenous LXRxcex1 alleles. The mammal of the present invention may a mouse, rat, hamster, guinea pig, rabbit, cow, and sheep, or other suitable organism.
In particular embodiments, either one or both non-functional LXRxcex1 allele may contain (a) an interruption in the LXRxcex1 coding sequence, (b) a nonsense mutation in the LXRxcex1 coding sequence or (c) a deletion of LXRxcex1 coding sequence. Alternatively, the non-functional LXRxcex1 allele may result from an alteration in the regulatory region of the LXRxcex1 gene. In a particular embodiment, substitution of the endogenous an inducible/repressable promoter for the endogenous LXRxcex1 promoter may be utilized. Cells of the transgenic mammal may further comprise an exogenous selectable marker gene under the control of a promoter active in at least one mammalian cell type.
Another aspect of the present invention provides a method for screening an RXR agonist or LXRxcex1 agonist candidate substance for the ability to increase bile acid synthesis comprising: 1) providing a cell, 2) contacting the cell with the candidate substance; and 3) monitoring a bile acid-related phenotype of the cell, wherein an increase in the bile acid-related phenotype in the cell treated with the candidate substance, as compared to a similar cell not treated with the candidate substance, indicates that the candidate substance increases bile acid synthesis. In one embodiment of the present invention the cell is a liver cell. The bile acid-related phenotype may be expression of a gene involved in bile acid synthesis, for example, Cyp7a. The candidate substance may be an RXR agonist, more particularly a rexinoid.
Also contemplated in the present invention is a method for screening a candidate substance for the ability to reduce cholesterol levels in a mammal comprising: 1) providing a non-human transgenic mammal, the cells of which comprise at least one non-functional endogenous LXRxcex1 allele; 2) treating the mammal with the candidate substance; and 3) monitoring a cholesterol-related phenotype in the mammal, wherein a reduction in the cholesterol-related phenotype in mammals treated with the candidate substance, as compared to a similar mammal not treated with the candidate substance, indicates that the candidate substance reduces cholesterol levels. The mammal may be selected from the group consisting of mouse, rat, hamster, guinea pig, rabbit, cow, and sheep. The cells of the mammal may comprise two non-functional endogenous LXRxcex1 alleles.
The phenotype may be cholesterol absorption, circulating cholesterol, hepatic cholesterol, hepatomegaly, atherosclerosis, cardiac failure, cardiac (atrophy/hypertrophy), activity level, survival, cancer, reproduction, immune function, skin disease, cognitive function, and adrenal function. The mammal may be maintained on a high cholesterol diet. The mammal may further be treated with an agent that blocks cholesterol biosynthesis.
Also contemplated is a method for screening a candidate substance for the ability to increase bile acid synthesis in a mammal comprising: 1) providing a non-human transgenic mammal, the cells of which comprise at least one non-functional endogenous LXRxcex1 allele; 2) treating the mammal with the candidate substance; and 3) monitoring a bile acid-related phenotype in said mammal wherein an increase in the bile acid-related phenotype in mammals treated with the candidate substance, as compared to a similar mammal not treated with the candidate substance, indicates that the candidate substance increases bile acid synthesis. The mammal may be selected from the group consisting of mouse; rat, hamster, guinea pig, rabbit, cow, and sheep. The bile acid-related phenotype may be selected from the group consisting of cholesterol level, Cyp7a synthesis, fecal bile acid excretion, bile acid pool size and bile acid composition.
Another aspect of the present invention is a method for screening a rexinoid for the ability to inhibit cholesterol absorption by an intestinal cell comprising: 1) providing an intestinal cell; 2) treating the cell with the rexinoid; and 3) monitoring cholesterol absorption by the cell, wherein a reduction in cholesterol absorption by the cell treated with the rexinoid, as compared to a similar cell not treated with the rexinoid, indicates that the rexinoid is an inhibitor of cholesterol absorption. The cell may be a duodenal cell, optionally located in a mammal. The method may further comprise comparing the effect of the candidate substance on cholesterol absorption on a cell comprising one or two non-functional endogenous LXRxcex1 alleles.
Another aspect of the present invention is a method of reducing cholesterol levels in a mammal comprising the step of treating the mammal with an RXR agonist. The agonist may be a rexinoid. The method may further comprise treating the mammal with an agent that inhibits cholesterol biosynthesis, for example, an HMG CoA reductase inhibitor. The mammal may be a human. The method may further comprise stimulating bile acid synthesis in the mammal or reducing cholesterol intake by the mammal.
In yet another aspect of the present invention, there is provide a method for inhibiting cholesterol absorption in a mammal comprising treating the mammal with an RXR agonist. The agonist may be a rexinoid and the mammal may be human
The present invention further contemplates a transgenic cell which comprises at least one non-functional endogenous LXRxcex1 allele. Additionally, the transgenic cell may comprises two non-functional endogenous LXRxcex1 alleles.
Also contemplated is a rexinoid compound that inhibits cholesterol absorption, identified by a process comprising: 1) providing an intestinal cell; 2) treating the cell with a rexinoid; and 3) monitoring cholesterol absorption by the cell, wherein a reduction in cholesterol absorption by the cell treated with the rexinoid, as compared to a similar cell not treated with the rexinoid, identified the rexinoid as an inhibitor of cholesterol absorption.
Additionally, the present invention provides a rexinoid compound that inhibits cholesterol absorption, produced by a process comprising: 1) providing an intestinal cell; 2) treating the cell with a rexinoid; 3) monitoring cholesterol absorption by the cell, wherein a reduction in cholesterol absorption by the cell treated with said rexinoid, as compared to a similar cell not treated with a rexinoid, identified the rexinoid as an inhibitor of cholesterol absorption; and 4) producing said rexinoid compound.
The present invention also provides a method of screening for a modulator of ABC1 expression comprising: 1) providing a cell expressing an RXR; 2) contacting said cell with a rexinoid and a candidate substance; and 3) determining the expression of ABC1 in said cell, wherein a change in expression of ABC1, as compared to a cell of step (b), indicates that said candidate substance is a modulator of ABC1 expression. Screening for a modulator of ABC1 expression further comprises the step of determining the expression of ABC1 in a cell expressing RXR in the absence of said candidate substance. ABC1 expression is measured by RNA analysis, such as Northern analysis or PCR. Alternatively, ABC1 expression is measured by protein analysis, such as ELISA or Western blot. In preferred embodiments, screening for a modulator of ABC1 expression in a cell comprises an exogenous marker cassette comprising a polynucleotide encoding a screenable marker operably linked to an ABC1 promoter region. In preferred embodiments, the screenable marker is an esterase, phosphatase, protease, green flourescent protein, luciferase, chloramphenicol acetyl transferase, xcex2-galactosidase. xcex2-glucuronidase or a drug resistance marker. Preferred cells for screening for a modulator of ABC1 expression are cells expressing an RXR such as intestinal cells, preferably of duodenal or jejunal origin. Screening for a modulator of ABC1 expression may be done in vivo.
The present invention also provides for a method of making a modulator of ABC1 expression comprising: 1) providing a cell expressing an RXR; 2) contacting said cell with a rexinoid and a candidate substance; 3) determining the expression of ABC1 in said cell, wherein a change in expression of ABC1, as compared to a cell of step (b), indicates that said candidate substance is a modulator of ABC1 expression; and 4) making said modulator.